Overall Program-Abstract The formation of inhibitory antibodies to infused factor VIII (FVIII) remains one of the most challenging complications of protein replacement therapy in hemophilia A (HA) patients and it is associated with increase morbidity and mortality. This U54 application assembles a cross-disciplinary team of investigators with appropriate track records, research interests and synergistic expertise to address unanswered mechanistic questions related to FVIII immunogenicity. Our innovative scientific program is well-integrated, tests new hypotheses and brings novel innovative technologies and investigators from a wide range of background to better understand FVIII immunogenicity Project 1 (Arruda/Milone): Characterization of the functional repertoire and ontogeny of FVIII humoral response across species. Studies using immunoproteomics and genomics to help rigorously determine the ontogeny of the inhibitor producing cells in HA patients. They will define the B cell repertoires responsible for the inhibitors in these patients and in longitudinal studies in HA dogs with inhibitors. Moreover, the will define the emerging role of B cell survival cytokine in the context of FVIII inhibitors. Project 2 (Herzog): In vivo Mechanism of Immune Response to Factor VIII. Utilizing innovative strategies, including in vivo visualization techniques to define which antigen presenting cells (APCs) are required for MHC II presentation to CD4+ T cells how these APCs interact to prime FVIII-specific CD4+ T cells, which subsets of CD4+ T cells are induced to promote B cell activation, and how innate immune signaling and the microbiome may alter these events. Project 3 (Lillicrap): Influence of the host microbiome on the mechanism of FVIII immunogenicity. Innovative preliminary observations linking elements of the gut microbiome in determining the FVIII-specific immune response. Employing animal studies to investigate the modulatory role of the host gut microbiome on the immune response to FVIII. This novel line of investigation is proposed to reveal a critical link between environmental factors and variability in the development of inhibitory antibodies to FVIII. Project 4 (Camire/Krishnaswamy): Factor VIII Immunogenicity-Biology and Structure. This project centers on the role of various molecular species relevant to the biological life cycle of FVIII in regulating the immune response and inhibitor development. Using biochemical and structural biological approaches to focus on the properties of FVIII driving the immune response. A major hypothesis to be pursued under this project relates to the role played by the interaction between FVIII and vWF as a modulator of inhibitor formation. The multi-pronged approach contained in this integrated proposal derives from the established areas of expertise of the participating investigators and provides a comprehensive investigation into the multiple biological mechanisms underlying the immunogenicity of FVIII.